Myeloid blood cells are a group of cells classified as white blood cells, including macrophages, dendritic cells, granulocytes, and so forth. Macrophages are the major cells that handle foreign substances in a living organism and have a role of defending the living organism from infectious diseases by phagocytosing and degrading infectious microorganisms or the like that have invaded the living organism. In addition, cell deaths occur daily in a large number in a living organism, and macrophages phagocytose and degrade debris thereof existing in the tissue of the living organism. In addition to these, macrophages also play an essential role in homeostatic maintenance of a living organism by processing various metabolites generated in the living organism by means of phagocytosis and degradation thereof. Additionally, it is recognized that macrophages often locally infiltrate malignant tumors. It is thought that there are cases in which macrophages locally existing in tumors attack the tumor cells, as well as cases in which they facilitate proliferation of the tumor cells. In the past, there have also been attempts to treat malignant tumors by utilizing the ability of macrophages to attack tumor cells.
Dendritic cells are cells that activate T lymphocytes by strongly stimulating them, and they are cells that regulate the immune response in living organisms. When infectious microorganisms invade a living organism, the dendritic cells phagocytose the microorganisms, provide the T lymphocytes with antigenic substances derived therefrom, and induce an immune response by stimulating and activating antigen-specific T lymphocytes. There have been attempts to employ dendritic cells as cell vaccines in immunotherapy against cancers and infectious diseases by utilizing the ability of the dendritic cells to strongly stimulate T lymphocytes.
In order to utilize macrophages or dendritic cells as cell drugs, thereby achieving clinical effects, a large number of cells are required. In the case of macrophages, about 1010 to 1012 of cells are required while, in the case of dendritic cells, 108 to 109 of cells are required. The number of these cells that exist in a living organism is limited, and also, it is difficult to collect a large number of these cells from a tissue in a living organism. Thus, in order to realize cell therapy by means of macrophages or dendritic cells, it is necessary to establish a method which can stably supply such myeloid blood cells in a large number and at a lower cost.
Macrophages and dendritic cells are cells that play important roles in pathophysiology of cancer, immune-related diseases, metabolic diseases, vascular diseases, and so forth. In developing various pharmaceuticals to treat these diseases, it is necessary to assess effects of drugs on macrophages and dendritic cells. In order to compare effects of many types of pharmaceutical-candidate chemical substances under the same conditions, a method of supplying a large number of macrophages or dendritic cells, possessing uniform characteristics, is required.
Pluripotent stem cells, such as embryonic stem cells (ES cells) or artificial pluripotent stem cells (induced pluripotent stem cells, i.e., iPS cells), are cells that possess an ability to differentiate into various cells, and said cells also possess an ability to proliferate nearly unlimitedly. Meanwhile, methods of creating myeloid blood cells, which have a certain functional similarity with macrophages or dendritic cells existing in living organisms, from pluripotent stem cells have been reported (for example, see Patent Literature 1, and Non-Patent Literatures 1-9). Therefore, it may be theoretically possible to create a large number of myeloid blood cells by proliferating a large number of pluripotent stem cells and by then differentiating them by use of such differentiation induction methods. For example, Patent Literature 1 discloses a method of differentiating human embryonic stem cells into dendritic cells, including (A) a step of co-culturing the human embryonic stem cells and cells, possessing properties of inducing differentiation and proliferation of blood cells, to obtain a cell group A; (B) a step of co-culturing the cell group A obtained in the above step (A) and the cells, possessing properties of inducing differentiation and proliferation of blood cells, in the presence of a granulocyte-macrophage colony stimulating factor (GM-CSF) and a macrophage colony stimulating factor (M-CSF) to obtain a cell group B; and (C) a step of culturing the cell group B obtained in the above step (B) in the presence of GM-CSF and interleukin-4 (IL-4). However, including the differentiation method disclosed in Patent Literature 1, differentiation induction culturing methods that have been reported in the past require considerable effort and time (one month or longer), and therefore, the cost and time requirements are excessive for methods of creating myeloid blood cells for the purpose of using them in cell therapy. In addition, in the past, there has been no report of a method which allows myeloid blood cells, created through differentiation induction of pluripotent stem cells, to proliferate for an extended period of time (for one month or longer) and which makes it possible to create a large number of myeloid blood cells (for example, 105 times or more of the number of pluripotent stem cells used as the starting materials).
Meanwhile, there have been well-known methods of creating dendritic cells and macrophages from monocytes in human peripheral blood (cells which express CD14 molecules in the peripheral blood). Since about 20,000 to 50,000 monocytes exist in 1 mL of peripheral blood of healthy humans, it is possible to separate monocytes from human peripheral blood by use of an indicator of expression of CD 14 molecules and to thus create dendritic cells and macrophages by using them. However, as it is difficult to proliferate human peripheral monocytes through ex vivo culturing, 1010 monocytes are required to create 1010 dendritic cells or macrophages, and, to obtain such a number of monocytes, it is required to separate monocytes from about 20 L of peripheral blood. Accordingly, at present, in the case of creating dendritic cells for performing cell vaccine therapy against cancers, white-blood-cell separation by means of cell separation using a blood-component collection device (apheresis) and, additionally, separation of monocytes among the white blood cells have been performed. In addition, there has been a problem in which it is difficult to stably create dendritic cells because large differences exist among donors in terms of the number of monocytes in peripheral blood and the ability thereof to differentiate ex vivo.    Patent Literature 1: PCT International Publication No. WO 2008/056734    Non-Patent Literature 1: Fairchild, P. J, Brook, F A, Gardner, R L, Graca, L, Strong, V, Tone, Y, Tone, M, Nolan, K F, Waldmann, H.2000 Directed differentiation of dendritic cells from mouse embryonic stem cells. Curr Biol. 10:1515-1518.    Non-Patent Literature 2: Lindmark, H, Rosengren, B, Hurt-Cmejo, E, and Bruder, C E. 2004. Gene expression profiling shows that macrophages derived from mouse embryonic stem cells is an improved in vitro model for studies of vascular disease. Exp Cell Res 300:335-344.    Non-Patent Literature 3: Zhan, X, Dravid, G, Ye, Z, Hammond, H, Shamblott, M, Gearhart, J, and Cheng, L. 2004. Functional antigen-presenting leucytes derived from human embryonic stem cells in vitro. Lancet 364:163-171.    Non-Patent Literature 4: Slukvin, II, Vodyanik, M A, Thomson, J A, Gumenyuk, M. E, and Choi, K D. 2006. Directed differentiation of human embryonic stem cells into functional dendritic cells through the myeloid pathway. J Immunol 176:2924-2932.    Non-Patent Literature 5: Odegaard, J I, Vats, D, Zhang, L, Ricardo-Gonzalez, R, Smith, K L, Sykes D B, Kamps, M P, and Chawla, A. 2007. Quantitative expansion of ES cell-derived myeloid progenitors capable of differentiating into macrophages. J Leukoc Biol 81:711-719.    Non-Patent Literature 6: Su, Z, Frye, C, Bae, K M, Kelley, V, and Vieweg, J. 2008. Differentiation of human embryonic stem cells into immunostimulatory dendritic cells under feeder-free culture conditions. Clin Cancer Res 14:6207-6217.    Non-Patent Literature 7: Tseng, S Y, Nishimoto, K P, Silk, K M, Majumdar A S, Dawes, G N, Waldmann, H, Fairchild, P J, Lebkowski, J S, and Reddy, A. 2009. Generation of immunogenic dendritic cells from human embryonic stem cells without serum and feeder cells. Regen Med 4:513-526.    Non-Patent Literature 8: Senju S, Suemori H, Zembutsu H, Uemura Y, Hirata S, Fukuma D, Matsuyoshi H, Shimomura M, Haruta M, Fukushima S, Matsunaga Y, Katagiri T, Nakamura Y, Furuya M, Nakatsuji N, and Nishimura Y. Genetically manipulated human embryonic stem cell-derived dendritic cells with immune regulatory function. Stem cells 25:2720-2729, 2007.    Non-Patent Literature 9: Choi, K D, Vodyanik, M A, and Slukvin, II. 2009. Generation of mature human myelomonocytic cells through expansion and differentiation of pluripotent stem cell-derived lin-CD34+CD43+CD45+progenitors. J Clin Invest 119:2818-2829.